Increased expression of α5 subunit and adhesion of JAK2V617F+ megakaryocytes to fibronectin results in megakaryocytosis. (A) Immunohistochemical staining of fibronectin (brown) in BM of C57BL/6J WT and JAK2V617F+ mice (26-week-old males). Top: whole-bone scanning (×0.4 original magnification). Inset squares: approximate location of images shown in bottom panels (×20 original magnification; scale bars represent 50 μm). (B) Percentage of CD41+CD42d− and CD41+CD42d+ megakaryocytes in BM of WT (n = 6 females and 4 males, 15 weeks old) and JAK2V617F+ mice (n = 5 females and 4 males, 15 weeks old). The differential effect persisted in the 2 sexes. (C) Cell surface expression of α5 (HMα5-1 antibody) and β1 (Ha2/5 antibody) subunits on CD41+ megakaryocytes differentiated in vitro. Data represent 2 independent experiments from WT (n = 4, 21-week-old males and n = 3, 18-week-old females) and JAK2V617F+ mice (n = 3, 21-week-old males and n = 4, 18-week-old females). Note that y-axis does not start at 0. (D) Adhesion assay of in vitro-differentiated megakaryocytes on plates coated with control BSA or FN, treated with an inhibitory antibody against α5 subunit (HMα5-1) or with a hamster IgG isotype control. Data represent 2 independent experiments, from WT (n = 2, 13-week-old and n = 2, 21-week-old males) and JAK2V617F+ mice (n = 4, 13-week-old and n = 3, 21-week-old males). (E) Cell surface expression of extended conformation β1 subunit (9EG7 antibody) in CD41+ megakaryocytes differentiated in vitro from WT and JAK2V617F+ mice (n = 5 each, 14-week-old males). (F) Similar analysis as in panel E, with megakaryocytes treated with an inhibitory antibody against α5 subunit (HMα5-1) or with a hamster IgG isotype control during in vitro differentiation on BSA or FN-coated plates. Data represent WT (n = 4, 18-week-old females) and JAK2V617F+ mice (n = 3, 18-week-old females). (G) Percentage of CD41+CD42d− and CD41+CD42d+ megakaryocytes in BM cultured under conditions as in panel F. Representative data from 1 of 2 independent experiments showing similar tendency are shown, from WT (n = 3, 10-week-old females) and JAK2V617F+ (n = 4, 10-week-old females). (H) Representative bright-field images of megakaryocytes differentiated in vitro in culture conditions shown in panel G. Arrows indicate some examples of megakaryocytes (×10 original magnification; scale bars represent 100 μm). Data are expressed as mean ± standard deviation; ns, not significant. *P < .05, **P < .01, ***P < .001, ****P < .0001.